We have previously demonstrated that in a rat model of trauma-hemorrhage (T-H) glucosamine administration during resuscitation improved cardiac function, reduced circulating levels of inflammatory cytokines and increased tissue levels of O-linked N-acetylglucosamine (GlcNAc) on proteins. The mechanism(s) by which glucosamine mediated its protective effect were not determined; therefore, the goal of this study was to test the hypothesis that glucosamine treatment attenuated activation of the Nuclear Factor-B (NF-B) signaling pathway in the heart via an increase in protein O-GlcNAc levels. Fasted male rats were subjected to T-H by bleeding to a mean arterial blood pressure 40 mmHg for 90 min followed by resuscitation. Glucosamine treatment during resuscitation significantly attenuated the T-H induced increase in cardiac levels of TNF- and IL-6 mRNA, IB- phosphorylation, nuclear NF-B, NF-B DNA binding activity, ICAM-1 and MPO activity. LPS (2µg/ml) increased levels of IB- phosphorylation, TNF-, ICAM1 and nuclear NF-B in primary cultured cardiomyocytes, which was significant attenuated by glucosamine treatment and overexpression of O-GlcNAc transferase (OGT); both interventions also significantly increased O-GlcNAc levels. In contrast, transfection of NRVMs with OGT siRNA decreased OGT and O-GlcNAc levels and enhanced LPS-induced increase in IB- phosphorylation. Glucosamine treatment of macrophage cell line RAW 264.7 also increased O-GlcNAc levels and attenuated the LPS induced activation of NF-B. These results demonstrate that modulation of O-GlcNAc levels alters the response of cardiomyocytes to activation of NF-B pathway, which may contribute to the glucosamine-mediated improvement in cardiac function following hemorrhagic shock.